Osteolineage niche cells initiate hematopoietic stem cell mobilization

Recent studies have implicated bone-lining osteoblasts as important regulators of hematopoietic stem cell (HSC) self-renewal and differentiation; however, because much of the evidence supporting this notion derives from indirect in vivo experiments, which are unavoidably complicated by the presence of other cell types within the complex bone marrow milieu, the sufficiency of osteoblasts in modulating HSC activity has remained controversial. To address this, we prospectively isolated mouse osteoblasts, using a novel flow cytometry–based approach, and directly tested their activity as HSC niche cells and their role in cyclophosphamide/granulocyte colony-stimulating factor (G-CSF)–induced HSC proliferation and mobilization. We found that osteoblasts expand rapidly after cyclophosphamide/G-CSF treatment and exhibit phenotypic and functional changes that directly influence HSC proliferation and maintenance of reconstituting potential. Effects of mobilization on osteoblast number and function depend on the function of ataxia telangiectasia mutated (ATM), the product of the Atm gene, demonstrating a new role for ATM in stem cell niche activity. These studies demonstrate that signals from osteoblasts can directly initiate and modulate HSC proliferation in the context of mobilization. This work also establishes that direct interaction with osteolineage niche cells, in the absence of additional environmental inputs, is sufficient to modulate stem cell activity.     

Aging of hematopoietic stem cells is regulated by the stem cell niche

Adult stem cells provide the basis for regeneration of aging tissue. Their dual ability for self-renewal and multilineage differentiation is controlled by direct interaction with a specific microenvironment -- the so called "stem cell niche". Hematopoietic stem cells (HSC) reside in the bone marrow. It is still under debate if HSC can rejuvenate infinitively or if they do not possess "true" self-renewal and undergo replicative senescence such as any other somatic cell. Furthermore, the question arises to what extent age-related changes in HSC are due to intrinsic factors or regulated by external stimuli. There is growing evidence, that the stem cell niche is most important for the regulation of cellular aging in adult stem cells. It is the stem cell niche that (i) maintains HSC in a quiescent state that reduces DNA damage as well as replicative senescence, (ii) protects from radicals and toxic compounds, (iii) regulates cell intrinsic signal cascades and (iv) modulates gene expression and epigenetic modifications in HSC. Thus, the interplay with the stem cell niche controls HSC function including the aging process of the hematopoiesis.     

Aging of the hematopoietic stem cells niche

Homeostasis of the hematopoietic system has its roots in the maintenance of hematopoietic stem cells (HSCs) in the bone marrow (BM). HSCs change both phenotypically and functionally with physiological age. The alterations noted in aged HSCs are thought to be a consequence of both cell-intrinsic and extrinsic changes. We review here the age-related changes that the BM microenvironment exerts on HSCs.     

A molecular profile of a hematopoietic stem cell niche

The hematopoietic microenvironment provides a complex molecular milieu that regulates the self-renewal and differentiation activities of stem cells. We have characterized a stem cell supportive stromal cell line, AFT024, that was derived from murine fetal liver. Highly purified in vivo transplantable mouse stem cells are maintained in AFT024 cultures at input levels, whereas other primitive progenitors are expanded. In addition, human stem cells are very effectively supported by AFT024. We suggest that the AFT024 cell line represents a component of an in vivo stem cell niche. To determine the molecular signals elaborated in this niche, we undertook a functional genomics approach that combines extensive sequence mining of a subtracted cDNA library, high-density array hybridization and in-depth bioinformatic analyses. The data have been assembled into a biological process oriented database, and represent a molecular profile of a candidate stem cell niche.     

Regulation of hematopoietic stem cells by the niche

The quiescent state in the cell cycle is thought to be indispensable for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as niches, is critical for maintaining the stem cell properties of HSCs, including cell adhesion, survival, and cell division. Hematopoietic stem cells balance quiescence and cell division in the stem cell niche and also maintain the potential for long-term hematopoiesis. We have recently reported that HSCs expressing the receptor tyrosine kinase Tie2 are in the G0 phase and anti-apoptotic, and comprise a side-population (SP) of HSCs, which contacts osteoblasts (OBs), the source of the angiopoietin-1 (Ang-1) ligand for Tie2 in the bone marrow (BM) niche. Tie2/Ang-1 signaling occurs in interactions between HSCs and niche cells. The interaction of Tie2 with Ang-1 in vitro induces tight adhesion of HSCs to stromal cells and is sufficient to maintain the long-term blood-repopulating (LTR) activity of HSCs in vivo by preventing cell division. In addition, Ang-1 enhances the ability of HSCs to become quiescent and induces their adhesion to the bone surface in vivo, resulting in protection of the HSC compartment from stresses suppressing hematopoiesis. These data suggest that the Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in the adult BM niche. Ang-1 produced by OBs activates Tie2 on HSCs and promotes tight adhesion of HSCs to the niche, resulting in quiescence and enhanced survival of HSCs.     

Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals

The limited vessel-forming capacity of infused endothelial progenitor cells (EPCs) into patients with cardiovascular dysfunction may be related to a misunderstanding of the biologic potential of the cells. EPCs are generally identified by cell surface antigen expression or counting in a commercially available kit that identifies "endothelial cell colony-forming units" (CFU-ECs). However, the origin, proliferative potential, and differentiation capacity of CFU-ECs is controversial. In contrast, other EPCs with blood vessel-forming ability, termed endothelial colony-forming cells (ECFCs), have been isolated from human peripheral blood. We compared the function of CFU-ECs and ECFCs and determined that CFU-ECs are derived from the hematopoietic system using progenitor assays, and analysis of donor cells from polycythemia vera patients harboring a Janus kinase 2 V617F mutation in hematopoietic stem cell clones. Further, CFU-ECs possess myeloid progenitor cell activity, differentiate into phagocytic macrophages, and fail to form perfused vessels in vivo. In contrast, ECFCs are clonally distinct from CFU-ECs, display robust proliferative potential, and form perfused vessels in vivo. Thus, these studies establish that CFU-ECs are not EPCs and the role of these cells in angiogenesis must be re-examined prior to further clinical trials, whereas ECFCs may serve as a potential therapy for vascular regeneration.     

Bone and the hematopoietic niche: a tale of two stem cells

The revived interest in (hematopoietic) stem cell (HSC) niches has highlighted the role of multiple cellular players found in the bone environment. Initially focused on the role of osteoblasts and sinusoid endothelial cells, the quest for HSC niche cells has recently focused on a unique role for osteoprogenitor cells (skeletal stem cells, mesenchymal stem cells). Strongly validated by observations of HSC dysregulation dictated by the dysregulation of osteoprogenitors, the role of osteoprogenitors in the HSC niche integrates data from different studies into a unified view. As preosteoblastic, periendothelial cells residing at the sinusoid wall, skeletal progenitors reconcile the notions of "osteoblastic" and "sinusoidal" niches with one another. In addition, they bring into focus the cross-regulation of skeletal and hematopoietic physiology as rooted into the interplay of two stem cells (hematopoietic and skeletal) sharing a single niche. As direct regulators of hematopoietic space formation, sinusoid development, and hematopoietic function(s), as well as direct progenitors of positive and negative regulators of HSCs such as osteoblasts and adipocytes, skeletal progenitors have emerged as pivotal organizers of a complex, highly plastic niche. This development seems to represents an evolutionary advance over the deterministic stem cell niches found in archetypal invertebrate systems.     

The retinoblastoma tumor suppressor is a critical intrinsic regulator for hematopoietic stem and progenitor cells under stress

The retinoblastoma tumor suppressor protein (RB) plays important roles in the control of the cell division cycle. It is estimated that RB is dysfunctional/inactivated in up to 40% of human leukemias. The consequences of loss of RB on hematopoietic stem and progenitor cell (HSPC) function in vivo are incompletely understood. Here, we report that mice genetically deficient in Rb in all hematopoietic cells (Vav-Cre Rb knockout [KO] animals) showed altered contribution of distinct hematopoietic cell lineages to peripheral blood, bone marrow, and spleen; significantly increased extramedullary hematopoiesis in the spleen; and a 2-fold increase in the frequency of hematopoietic progenitor cells in peripheral blood. Upon competitive transplantation, HSPCs from Vav-Cre Rb KO mice contributed with an at least 4- to 6-fold less efficiency to hematopoiesis compared with control cells. HSPCs deficient in Rb presented with impaired cell-cycle exit upon stress-induced proliferation, which correlated with impaired function. In summary, Rb is critical for hematopoietic stem and progenitor cell function, localization, and differentiation.     

Genetic regulation of primitive hematopoietic stem cell senescence

OBJECTIVE: To define effects of strain on PHSC (primitive hematopoietic stem cells) senescence (decline in function with age) in vivo, and to map a locus that regulates PHSC senescence. MATERIALS AND METHODS: Long-term function and self-renewal were compared in bone marrow cells (BMC) from old and young mice of three strains: BALB/cBy (BALB), DBA/2 (D2) and C57BL/6 (B6), using competitive repopulation and serial transplantation in vivo. BMC from each old or young donor were mixed with standard doses of congenic, genetically marked BMC and transplanted into lethally recipients. Percentages of donor-type erythrocytes and lymphocytes in the recipients determined the functional ability of donor PHSC relative to the standard, where one repopulating unit (RU) of donor BMC equals the repopulating ability of 100,000 standard competitor BMC. Using similar techniques, repopulating abilities of old and young recombinant inbred (RI) donors of 12 strains derived from BALB and B6 were compared in NK-depleted BALBxB6 Fl recipients to map a locus that appears to have a major role in PHSC senescence. RESULTS: PHSC function declined about 2 fold with age in BALB and D2 BMC, and increased more than 2-fold with age in B6 BMC, with all old/young strain differences significant, p<.01. Ten months after serial transplantation, young B6, BALB, and D2 PHSC had self-renewed 1.6-, 4.2-, and 3.2-fold better than old, with BALB and D2 old/young differences p<.01. Young B6 PHSC self-renewed 1.9- and 2.9-fold better than young BALB and D2 PHSC. The PHSC senescence phenotypes (old/young RU ratios) for 12 CXB RI strains suggested a genetic linkage to D12Nyul7 on Chromosome 12. CONCLUSION: PHSC senescence is genetically regulated, and is much delayed in the B6 strain compared to the BALB and D2 strains. A locus on Chromosome 12 may regulate PHSC senescence.     

Impact of modeled microgravity on migration, differentiation, and cell cycle control of primitive human hematopoietic progenitor cells

OBJECTIVE: Migration, proliferation, and differentiation of bone marrow (BM) hematopoietic stem cells (HSC) are important factors in maintaining hematopoietic homeostasis. Homeostatic control of erythrocytes and lymphocytes is perturbed in humans exposed to microgravity (micro-g), resulting in space flight-induced anemia and immunosuppression. We sought to determine whether any of these anomalies can be explained by micro-g-induced changes in migration, proliferation, and differentiation of human BM CD34+ cells, and whether such changes can begin to explain any of the shifts in hematopoietic homeostasis observed in astronauts. MATERIALS AND METHODS: BM CD34+ cells were cultured in modeled micro-g (mmicro-g) using NASA's rotating wall vessels (RWV), or in control cultures at earth gravity for 2 to 18 days. Cells were harvested at different times and CD34+ cells assessed for migration potential, cell-cycle kinetics and regulatory proteins, and maturation status. RESULTS: Culture of BM CD34+ cells in RWV for 2 to 3 days resulted in a significant reduction of stromal cell-derived factor 1 (SDF-1alpha)-directed migration, which correlated with decreased expression of F-actin. Modeled micro-g induced alterations in cell-cycle kinetics that were characterized by prolonged S phase and reduced cyclin A expression. Differentiation of primitive CD34+ cells cultured for 14 to 18 days in RWV favored myeloid cell development at the expense of erythroid development, which was significantly reduced compared to controls. CONCLUSIONS: These results illustrate that mmicro-g significantly inhibits the migration potential, cell-cycle progression, and differentiation patterns of primitive BM CD34+ cells, which may contribute to some of the hematologic abnormalities observed in humans during space flight.     

Co-culture of hematopoietic stem cells with mesenchymal stem cells increases VCAM-1-dependent migration of primitive hematopoietic stem cells

Hematopoietic stem cells (HSC) lose their capacity for engraftment during ex vivo cytokine expansion. It has been shown that mesenchymal stem cells (MSC) improve HSC transplantability; however, the molecular mechanisms responsible for this effect have not yet been completely elucidated. This paper reports that expanding HSC in co-culture with MSC enhances a vascular cell adhesion molecule (VCAM-1)-dependent pro-migratory phenotype. MSC did not regulate the HSC expression of CD49d (VCAM-1 counter-receptor molecule), but did decrease the cytokine-induced HSC VCAM-1-mediated pro-adhesive phenotype. Co-culture with MSC reduced the expression of the inactive conformation of lymphocyte function-associated antigen (LFA-1) at the HSC uropod, and induced higher expression of an LFA-1 activation epitope. Interestingly, VCAM-1-dependent HSC migration was modulated by targeting this LFA-1 high affinity form, suggesting integrin cross-regulation. VCAM-1-mediated HSC transmigration appeared to favor the more primitive HSC immunophenotype. Our results suggested that co-culture with MSC improved VCAM-1-dependent migration of primitive HSC, which was affected in ex vivo cytokine-expanded HSCs by a mechanism involving LFA-1 modulation.     

Hierarchical organization and regulation of the hematopoietic stem cell osteoblastic niche

Hematopoietic stem cells (HSCs) are characterized with long-term self-renewal and multi-lineage differentiation. HSCs preferentially reside in osteoblastic niche that is hierarchically organized so as to exactly regulate HSCs properties and maintain organism hematopoietic homeostasis. Hematopoietic stem cell–mesenchymal stem cell pairings share stem cell niches in adult bone marrow under immune–humoral–neural regulation. Here, we review the hierarchical HSC osteoblastic niche in terms of its localization, composition, function, and multi-layer regulation systems.